Cholera toxin (CT), responsible for the diarrhea caused by Vibrio cholerae, is also a potent mucosal vaccine adjuvant. After the pentameric B subunit of CT binds the GM1 ganglioside receptor, CT enters the cell and its catalytic A subunit activates Gs to induce adenosine 3, 5-monophosphate (cAMP) production by adenylate cyclase. In intestinal epithelial cells, cAMP induces chloride secretion, leading to the massive luminal fluid secretion that characterizes the diarrhea of cholera. However, the mechanisms by which CT induces a mucosal immune response are less well understood. Our studies showed that CT activates dendritic cells (DC) by cAMP-dependent mechanisms to drive differentiation of naive T cells into IL-17-producing Th17 cells in vitro and in vivo. This differentiation was independent of IL-6, and microarray analysis identified novel pathways involved in Th17 differentiation. Th17 cells, whose differentiation has previously been established to be dependent on IL-6 and TGF-beta, are a recently identified subset of CD4+ T cells that play an important role in autoimmunity as well as in the control of certain extracellular pathogens. The involvement of cAMP and other novel pathways highlights the potential role of these signaling pathways in determining T cell differentiation. These insights into the mechanisms controlling differentiation of this important subset of T cells also identify putative new targets to beneficially modulate immune responses involved in infections and autoimmunity.